Despite many years of research and despite great advances in our understanding of operon-specific regulation, we still do not have a clear view of how bacteria sense general nutrient insufficiency and how they integrate the multiple nutritional signals that they are constantly exposed to. These mechanisms are responsible for the remarkable ability of bacteria to adapt to constantly changing environments and influence the interactions, both symbiotic and parasitic, of bacteria with host cells and host organisms. In gram-negative bacteria, the leucine-responsive protein (Lrp) plays an important role in governing the expression of a wide variety of cellular functions in response to deprivation of leucine or certain other amino acids. In gram-positive bacteria, such as Bacillus subtilis, CodY protein seems to play an analogous role, even though CodY and Lrp are unrelated proteins. Prior work revealed that CodY has the unusual property of responding to two different, critical metabolites, GTP and isoleucine (or valine). CodY represses, directly or indirectly, the expression of hundreds of genes, most of which encode proteins that allow cells to adapt to limited nutritional resources. In collaboration with Dr. A. J. Wilkinson, the structure of CodY has been determined in its ligand-bound and ligand-free states, leading to a molecular model for the mechanism by which interaction with its ligands makes CodY an effective regulator. CodY has also been found to act as a positive regulator of genes of central metabolism. As a result, the CodY regulon overlaps in interesting and complex ways with other globally controlled regulons. Most CodY homologs are found in low G+C gram-positive bacteria, including major pathogens. Preliminary results indicate that CodY in such pathogens contributes in important ways to the control of virulence gene expression. The goals of this proposal are to determine how B. subtilis CodY structure and function intersect, to reveal the overall physiological role of CodY, to assess the effects of constitutive CodY activity, and to extend our understanding of the function of CodY by determining its role in the growth, adaptation and virulence of Staphylococcus aureus. PUBLIC HEALTH RELEVANCE: The CodY protein has been found to regulate the expression of many bacterial genes, including genes that encode toxins and other virulence factors. This proposal seeks to understand the physiological role of CodY in the life of the bacterium and to uncover the molecular mechanism by which CodY represses or activates gene expression.